Apparatus for using optical tweezers to manipulate materials

ABSTRACT

A method and apparatus for control of optical trap arrays and formation of particle arrays using light that is in the visible portion of the spectrum. The method and apparatus provides a laser and a time variable diffractive optical element to allow dynamic control of optical trap arrays and consequent control of particle arrays and also the ability to manipulate singular objects using a plurality of optical traps. By avoiding wavelengths associated with strong absorption in the underlying material, creating optical traps with a continuous-wave laser, optimizing the efficiency of individual traps, and trapping extended samples at multiple points, the rate of deleterious nonlinear optical processes can be minimized.

This invention was made with U.S. Government support under Contract No.DMR-9730189 awarded by the National Science Foundation, through theMRSEC Program of the National Science Foundation under Award No.DMR-9808595, and through a GAANN fellowship from the Department ofEducation. The U.S. Government also has certain rights to the invention.

The present invention is directed generally to a method and apparatusfor control of optical traps or optical tweezers. More particularly, theinvention is directed to optical tweezers formed using visible lightthat can be used to manipulate a variety of light sensitive materials,such as living biological materials, without substantial damage ordeleterious effects upon the material being investigated or manipulated.

It is known to construct optical tweezers using optical gradient forcesfrom a single beam of light to manipulate the position of a smalldielectric particle immersed in a fluid medium whose refractive index issmaller than that of the particle. The optical tweezer technique hasbeen generalized to enable manipulation of reflecting, absorbing and lowdielectric constant particles as well.

Some systems have therefore been developed which can manipulate a singleparticle by using a single beam of light to generate a single opticaltrap. To manipulate multiple particles with such systems, multiple beamsof light must be employed. The difficulty of creating extendedmultiple-beam traps using conventional optical tweezer methodologyinhibits their use in many potential commercial applications such as theinspection of biological materials generally, and also the fabricationand manipulation of nanocomposite materials including electronic,photonic and opto-electronic devices, chemical sensor arrays for use inchemical and biological assays, and holographic and computer storagematrices.

An optical tweezer uses forces exerted by an intense and tightly focusedbeam of light to trap and manipulate dielectric particles, typically influid media. Prior descriptions of optical tweezers emphasized theirpotential utility for biological applications such as capturing cells,or their components, for research, diagnostic evaluation, and eventherapeutic purposes. These same reports also emphasized the inherentand persistent occurrence of damage or changes caused by opticaltrapping methods when using visible light. In particular, it has beenobserved that green light of wavelength λ=514.5 nm from an Ar ion laserhas caused various deleterious effects on biological material: red bloodcells literally explode, the chloroplasts of green plant cells weredestroyed, and the continued application of a green laser light hascaused the death of trapped ciliated bacteria. Damage from the greenlaser light in the first two examples clearly resulted from strongabsorption of green light by hemoglobin and chlorophyll, respectively,leading to rapid heating and catastrophic destruction. The mechanism ofthe third type of damage, dubbed “opticution” by those in the art, wasnot immediately obvious. Subsequent studies have identifiedoptically-induced mutagensis to be a likely mechanism for the cells'death by virtue of the use of optical tweezers.

Considerably less damage to biological materials was observed whencomparable materials were optically trapped with infrared light from aNd:YAG laser operating at λ=1064 nm. Largely on the basis of these andsimilar early observations with a single optical tweezer, researcherscame to the conclusion that infrared illumination is operationallysuperior to visible illumination for optically trapping biologicalmaterials. That is, use of infrared light did not cause any apparentdeleterious effect upon biological material.

Laser-induced damage can be desirable, however, in specialcircumstances. For example, pulsed optical tweezers operating at λ=532nm have been singled out for their ability to cut biological materials,such as chromosomes. Optical tweezers used in this way are known asoptical scissors or optical scalpels. Even so, the prospects fornondestructively trapping biological materials with visible light hadpreviously been considered by those in the art to be an unacceptablemethod of optical trapping and manipulation due to the well documentedand accepted deleterious effect on biological material.

It is therefore an object of the invention to provide an improved methodand system for using at least one optical trap from light in the visibleor ultraviolet portion of the spectrum.

It is also an object of the invention to provide a novel method andapparatus for control of visible light optical traps that has anincreased level of efficiency, effectiveness and safety for use.

It is yet another object of the invention to provide a novel method andapparatus for control of visible light optical traps that is relativelysimple to align by virtue of using light visible to the human eye.

It is still a further object of the invention to provide a novel methodand apparatus for control of visible light optical traps where localizedregions can be accurately trapped.

It is an additional object of the invention to provide a novel methodand apparatus for control of visible light optical traps wherein thesamples being manipulated are not overly heated or otherwise altered dueto light absorption.

It is yet a further object of the invention to provide a novel methodand apparatus for control of optical traps wherein the optical trapshave highly improved tracking accuracy.

It is an another object of the invention to provide a novel method andapparatus for using visible light for optical tweezers for use on anymaterial whose electronic, mechanical, chemical or biological state ishighly sensitive to optical tweezers having a high intensity lightpattern.

It is still another object of the invention to provide a novel methodand apparatus for control of optical traps with a variable power levelwhich provides efficient optical trapping but without alteration of thedesired chemical, biological, electronic or mechanical state of thematerial.

In accordance with the above objects, it has been discovered that damageor unwanted alterations inflicted by visible optical tweezers onbiological matter, and other materials sensitive to high intensitylight, can be reduced to acceptable, de minimis or even zero dimensionsand levels, in part through appropriate design of the optical trappingsystem and method. In addition, it is believed that wavelengths in theultraviolet can also be used for particular small size objects andparticular types of materials by taking advantage of the features ofthis invention. Consequently, such optical tweezers can have widespreadapplications in biological systems and other systems having lightsensitive materials and possess a number of advantages over infraredoptical tweezers.

Other objects, features and advantages of the present invention will bereadily apparent from the following description of the preferredembodiments thereof, taken in conjunction with the accompanying drawingsdescribed below wherein like elements have like numerals throughout.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a method and system which includes some conventionalfeatures for a single, steerable optical tweezer;

FIG. 2 illustrates a method and system which includes some conventionalfeatures for a single steerable optical tweezer;

FIG. 3. illustrates a method and system using a diffractive opticalelement;

FIG. 4 illustrates another method and system using a tilted opticalelement relative to an input light beam;

FIG. 5 illustrates a continuously translatable optical tweezer (trap)array using a diffractive optical element;

FIG. 6 illustrates a method and system for manipulating particles usingan optical tweezer array while also forming an image for viewing theoptical trap array;

FIG. 7A illustrates an image of a four by four array of optical tweezers(traps) using the optical system of FIG. 6; and FIG. 7B illustrates animage of one micrometer diameter silica spheres suspended in water bythe optical tweezers of FIG. 7A immediately after the trappingillumination has been extinguished, but before the spheres have diffusedaway;

FIG. 8 illustrates a method and system for manipulating particles usingan optical tweezer away-while also accelerating the filling of opticaltraps;

FIGS. 9A-9D illustrate the use of optical trap control methodologywherein the optical traps are formed by a holographic diffractiveoptical element; and

FIG. 10 illustrates the use of optical trap control methodology whereina microscope images the particles and a personal computer is used toidentify the particles and calculate a phase only hologram to trap theparticles.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

In order to best understand the improvement of the invention, FIGS. 1and 2 illustrate several methods and systems which include someconventional features. In an optical tweezer system 10 of FIG. 1,optical gradient forces arise from use of a single beam of light 12 tocontrollably manipulate a small dielectric particle 14 dispersed in amedium 16 whose index of refraction, n_(m), is smaller than that of theparticle 14. The fundamental nature of the optical gradient forces iswell known, and also it is understood that the principle has beengeneralized to allow manipulation of reflecting, absorbing and lowdielectric constant particles as well. Any of these techniques can beimplemented in the context of the invention improvements describedhereinafter and will be encompassed by use of the terminology opticaltweezer, optical trap and optical gradient force trap hereinafter.

The optical tweezer system 10 is applied by using a light beam 12 (suchas a laser beam or other very high intensity light sources) capable ofapplying the necessary forces needed to carry out the optical trappingeffect needed to manipulate a particle. The objective of a conventionalform of the optical tweezer 10 is to project one or more shaped beams oflight into the center of a back aperture 24 of a converging opticalelement (such as an objective lens 20). As noted in FIG. 1 the lightbeam 12 has a width “w” and having an input angle Ø relative to anoptical axis 22. The light beam 12 is input to a back aperture 24 of theobjective lens 20 and output from a front aperture 26 substantiallyconverging to a focal point 28 in focal plane 30 of imaging volume 32with the focal point 28 coinciding with an optical trap 33. In general,any focusing optical system can form the basis for the optical tweezersystem 10.

In the case of the light beam 12 being a collimated laser beam andhaving its axis coincident with the optical axis 22, the light beam 12enters the back aperture 24 of the objective lens 20 and is brought to afocus in the imaging volume 32 at the center point c of the objectivelens focal plane 30. When the axis of the light beam 12 is displaced bythe angle Ø with respect to the optical axis 22, beam axis 31 and theoptical axis 22 coincide at the center point B of the back aperture 12.This displacement enables translation of the optical trap across thefield of view by an amount that depends on the angular magnification ofthe objective lens 20. The two variables, angular displacement Ø andvarying convergence of the light beam 12, can be used to form theoptical trap at selected positions within the imaging volume 32. Amultiple number of the optical traps 33 can be arranged in differentlocations provided that multiple beams of light 12 are applied to theback aperture 24 at the different angles Ø and with differing degrees ofcollimation.

In order to carry out optical trapping in three dimensions, opticalgradient forces created on the particle to be trapped must exceed otherradiation pressures arising from light scattering and absorption. Ingeneral this necessitates having the wave front of the light beam 12 tohave an appropriate shape at the back aperture 24. For example, for aGaussian TEM_(oo) input laser beam, the beam diameter w shouldsubstantially coincide with the diameter of the back aperture 24. Formore general beam profiles (such as Gauss-Laguerre) comparableconditions can be formulated.

In another system in FIG. 2 which includes some conventional features,the optical tweezer system 10 can translate the optical trap 33 acrossthe field of view of the objective lens 20. A telescope 34 isconstructed of lenses L1 and L2 which establishes a point A which isoptically conjugate to the center point B in the prior art system ofFIG. 1. In the system of FIG. 2 the light beam 12 passing through thepoint A also passes through the point B and thus meets the basicrequirements for performing as the optical tweezer system 10. The degreeof collimation is preserved by positioning the lenses L1 and L2 as shownin FIG. 2 to optimize the transfer properties of the telescope 34. Inaddition, the magnification of the telescope 34 can be chosen tooptimize angular displacement of the light beam 12 and its width w inthe plane of the back aperture 24 of the objective lens 20. As statedhereinbefore, in general several of the light beams 12 can be used toform several associated optical traps. Such multiple beams 12 can becreated from multiple independent input beams or from a single beammanipulated by conventional reflective and/or refractive opticalelements.

In one preferred embodiment of an overall optical manipulation systemshown in FIG. 3, arbitrary arrays of optical traps can be formed. Adiffractive optical element 40 is disposed substantially in a plane 42conjugate to back aperture 24 of the objective lens 20. Note that only asingle diffracted output beam 44 is shown for clarity, but it should beunderstood that a plurality of such beams 44 can be created by thediffractive optical element 40. The input light beam 12 incident on thediffractive optical element 40 is split into a pattern of the outputbeam 44 characteristic of the nature of the diffractive optical element40, each of which emanates from the point A. Thus the output beams 44also pass through the point B as a consequence of the downstream opticalelements described hereinbefore.

The diffractive optical element 40 of FIG. 3 is shown as being normal tothe input light beam 12, but many other arrangements are possible. Forexample, in FIG. 4 the light beam 12 arrives at an oblique angle βrelative to the optic axis 22 and not at a normal to the diffractiveoptical element 40. In this embodiment, the diffracted beams 44emanating from point A will form optical traps 50 in focal plane 52 ofthe imaging volume 32 (seen best in FIG. 1). In this arrangement of theoptical tweezer system 10 an undiffracted portion 54 of the input lightbeam 12 can be removed from the optical tweezer system 10. Thisconfiguration thus enables processing less background light and improvesefficiency and effectiveness of forming optical traps.

The diffractive optical element 40 can include computer generatedholograms which split the input light beam 12 into a preselected desiredpattern. Combining such holograms with the remainder of the opticalelements in FIGS. 3 and 4 enables creation of arbitrary arrays in whichthe diffractive optical element 40 is used to shape the wavefront ofeach diffracted beam independently. Therefore, the optical traps 50 canbe disposed not only in the focal plane 52 of the objective lens 20, butalso out of the focal plane 52 to form a three-dimensional arrangementof the optical traps 50.

In the optical tweezer system 10 of FIGS. 3 and 4, also included is afocusing optical element, such as the objective lens 20 (or other likefunctionally equivalent optical device, such as a Fresnel lens) toconverge the diffracted beam 44 to form the optical traps 50. Further,the telescope 34, or other equivalent transfer optics, creates a point Aconjugate to the center point B of the previous back aperture 24. Thediffractive optical element 40 is placed in a plane containing point A.

In another embodiment, arbitrary arrays of the optical traps 50 can becreated without use of the telescope 34. In such an embodiment thediffractive optical element 40 can be placed directly in the planecontaining point B.

In the optical tweezer system 10 either static or time dependentdiffractive optical elements 40 can be used. For a dynamic, or timedependent version, one can create time changing arrays of the opticaltraps 50 which can be part of a system utilizing such a feature. Inaddition, these dynamic optical elements 40 can be used to actively moveparticles and matrix media relative to one another. For example, thediffractive optical element 40 can be a liquid crystal phase arrayundergoing changes imprinted with computer-generated holographicpatterns.

In another embodiment illustrated in FIG. 5, a system can be constructedto carry out continuous translation of the optical tweezer trap 50. Agimbal mounted mirror 60 is placed with its center of rotation at pointA. The light beam 12 is incident on the surface of the mirror 60 and hasits axis passing through point A and will be projected to the backaperture 24. Tilting of the mirror 60 causes a change of the angle ofincidence of the light beam 12 relative to the mirror 60, and thisfeature can be used to translate the resulting optical trap 50. A secondtelescope 62 is formed from lenses L3 and L4 which creates a point A′which is conjugate to point A. The diffractive optical element 40 placedat point A′ now creates a pattern of diffracted beams 64, each of whichpasses through point A to form one of the tweezer traps 50 in an arrayof the optical tweezers system 10.

In operation of the embodiment of FIG. 5, the mirror 60 translates theentire tweezer array as a unit. This methodology is useful for preciselyaligning the optical tweezer array with a stationary substrate todynamically stiffen the optical trap 50 through small-amplitude rapidoscillatory displacements, as well as for any application requiring ageneral translation capability.

The array of the optical traps 50 also can be translated verticallyrelative to the sample stage (not shown) by moving the sample stage orby adjusting the telescope 34. In addition, the optical tweezer arraycan also be translated laterally relative to the sample by moving thesample stage. This feature would be particularly useful for large scalemovement beyond the range of the objective lens field of view.

In another embodiment shown in FIG. 6 the optical system is arranged topermit viewing images of particles trapped by the optical tweezers 10. Adichroic beamsplitter 70, or other equivalent optical beamsplitter, isinserted between the objective lens 20 and the optical train of theoptical tweezer system 10. In the illustrated embodiment thebeamsplitter 70 selectively reflects the wavelength of light used toform the optical tweezer array and transmits other wavelengths. Thus,the light beam 12 used to form the optical traps 50 is transmitted tothe back aperture 24 with high efficiency while light beam 66 used toform images can pass through to imaging optics (not shown).

An illustration of one application of an optical system is shown inFIGS. 7A and 7B. The diffractive optical element 40 is designed tointeract with the single light beam 12 to create a 4×4 array ofcollimated beams. A 100 mW frequency doubled diode-pumped Nd:YAG laseroperating at 532 nm provides a Gaussian TEM_(oo) form for the light beam12. In FIG. 7A the field of view is illuminated in part by laser lightbackscattered by sixteen silica spheres trapped in the array's sixteenprimary optical tweezers 10. The 1 μm diameter spheres are dispersed inwater and placed in a sample volume between a glass microscope slide anda 170 μm thick glass coverslip. The tweezer array is projected upwardthrough the coverslip and is positioned in a plane 8 μm above thecoverslip and more than 20 μm below the upper microscope slide. Thesilica spheres are stably trapped in three-dimensions in each of thesixteen optical tweezers 10.

In FIG. 7B is shown the optically-organized arrangement of spheres{fraction (1/30)} second after the optical tweezers 10 (traps) wereextinguished but before the spheres had time to diffuse away from thetrap site.

Adaptive Tweezer Mode

In various embodiments the basic optical trap modes describedhereinbefore can be used in various useful methodologies. Furthermore,other embodiments include apparati and systems which can be constructedto apply these methods to enhance operation and use of the opticaltraps. In particular, the optical traps can be controlled and modified,and various embodiments employing these features are describedhereinafter.

A variety of new uses and applications of optical traps can arise fromtime varying construction and dynamic change of optical trapconfiguration. In one form of the invention an array of optical trapscan be advantageously manipulated in the manner shown in FIG. 8. In theillustrated optical system 100, the diffractive optical element 102splits the collimated laser beam 104 into several (two or more) laserbeams 106 and 108. Each of the several laser beams 106 and 108 aretransferred into a separate optical trap in an object plane 118. Each ofthese several laser beams 106, 108 are transferred to the back aperture110 of the objective beam 112 by action of a conventional opticalarrangement, such as the telescope formed by the laser 114 and 116. Theobjective lens 112 focuses each of these several beams 106, 108. In apreferred form of the invention a movable knife edge 120 is disposed tobe movable into the path of the several laser beams 106, 108, therebyenabling selective blocking of any selected one(s) of the several laserbeams to selectively prevent formation of a portion of the opticaltraps. Such a methodology and structure enables construction of anydesired array of optical traps by use of appropriately designed knifeedges or apertured knife edge structure and like such structures.

An illustration of the use of such optical trap control methodology isshown in FIG. 9 wherein optical traps are formed by a holographic formof diffractive optical element 122. The movable knife edge 120 of FIG. 8can block all but one line 124 of its optical traps, and bysystematically moving the knife edge 120, each of the lines 124 can beestablished. This enables systematic filling of optical traps 132 withparticles 126. This methodology allows filling of the optical traps 132with a variety of different types of the particles 126 and also avoidsthe typical problem of the particles 126 tending to fill preferentiallythe outer portions of an array of optical traps. Such preferentialfilling can block filling of the inner optical traps. This controlledformation of the optical traps also permits precision formation andchange of optical trap arrangements.

In addition to exerting detailed control over filling of an array of theoptical traps 132, devices can be provided to accelerate filling of theoptical traps. For example, in FIG. 8 is shown a functional block 128indicative of a device to (1) output selected particles 126 (see FIG.10), (2) apply the particles 126 under pressure differential (throughelectrophoresis or electro-osmosis), (3) apply a temperature gradientand (4) translate the entire optical trap array through a suspensioncontaining the particles 126 in a manner like a fishing net.Experimentation has determined the particles 134 can be filled into theoptical traps 132 starting with a particle concentration of about 10⁻⁴μm⁻and a reasonable flow rate of about 100 μm/sec to fill one row of theline 124 or an array pattern in about one minute of time. A fullydeveloped array of the particles 126 can be made permanent bytransferring the array onto a substrate or by gelling the fluid which issuspending the particles. Such a procedure also can allow constructionof a large variety of different particle arrays and coupled arrays ofthe particles 126. Using the previously-described characteristics andfunctionalities of the optical traps 132, each of the particles 126 canalso be further interrogated, imaged and manipulated for operationaluses and investigative purposes.

In yet another embodiment the optical traps 132 can be dynamicallychanged responsive to a specific optical requirement, which can beeffected by use of a computer program with desired instructionalinformation such that one or more of the optical traps 132 can be usedto modify, remove, or add particles at various optical trap sites orallow various manipulations of a single object. Further, one or more ofthe optical traps 132 can be moved and their character changed (such aschanging the shape or strength of the trap) for dynamic manipulation ofany object, such as a cell of a plant or animal. This can beparticularly advantageous when manipulating a delicate structure or whenthere is need to perform complex manipulations of an object. Heretofore,such objects were handles by a single brute force trap which could causedamage to the object or not provide the degrees of freedom often neededto perform a desired function.

In addition, in another process the particles 126 can be dynamicallysorted by size. One can also image an array of the particles 126 in themanner shown in FIG. 10. A microscope 138 can image the particles 126,and a personal computer 140 can identify the particles 126 and calculatea phase only hologram 142 (for the diffractive optical element 144 ofFIG. 8) to trap said particles. A computer controlled spatial lightmodulator 143 can then implement the computer designed hologram 142 bycausing application of a pattern of phase modulations to the laser beam144. This can also be dynamically varied for any of a variety ofpurposes. The modified laser beam 148 (also see the several laser beams106, 108 in FIG. 8) are focused by the microscope to create an array ofthe optical traps 132 (also known as tweezers) which traps the particles126 on image screen 150. Each of the particles 126 can then beindividually manipulated to assemble a desired structure to sort theparticles 126 or to otherwise manipulate, inspect or alter the shape ofthe object of interest.

Use of Light in the Visible and UV Spectrum

In a preferred embodiment of the invention, visible light tweezers canbe used advantageously. In other forms of the invention, for particularsizes of materials matched to ultraviolet light or for uses which areless sensitive to ultraviolet light, the invention can also be expandedto wavelengths shorter than visible light, including ultraviolet light.Heretofore, tweezers for use in living biological material have beenformed from infrared light for the reasons described hereinbefore.Optical tweezers in general can damage biological systems through atleast three principal mechanisms: (1) mechanically disrupting physicalinterconnections; (2) heating; and (3) in the case of biomaterials,photochemical transformation of biomolecules (these are merely exemplarymechanisms and other mechanisms are possible). The first mechanismincludes processes such as drawing into the optical trap thephospholipids constituting a membrane and then expelling them asmicelles or vesicles. Such processes are inherent in the operation ofoptical tweezers and do not depend on the wavelength of light beingused. These destructive processes can be minimized by using the leastpossible and most efficient trapping force for a given application.Heating results from absorption of the optical trapping photons, astypified by the destruction of hemoglobin-rich red blood cells by greenlaser light. Most biological materials, however, are essentiallytransparent to visible light and in fact absorb more strongly in theinfrared. For example, water has an absorption coefficient of aboutμ_(∂)=3×10⁻⁴ cm⁻¹ at λ=500 nm (visible range) compared with μ_(∂)=0.1cm⁻¹ at λ=1 μm (infrared range). Infrared based optical traps shouldtherefore heat water some 300 times more efficiently than visible lightbased traps. The difference is far less pronounced for other componentsof biological systems. For example, most proteins and polysaccharideshave molar absorption coefficients of: μ_(∂)≈0.1 cm⁻¹ /M for visiblelight and μ_(∂)≈0.01 cm⁻¹/M for infrared radiation. Hemoglobin is anexception, with a comparatively enormous molar absorption coefficient ofμ_(∂)≈10⁴ cm⁻¹/M in the visible light part of the spectrum. In theabsence of such a strong absorption condition, visible light should leadto no worse heating than infrared, and indeed may well be preferablebecause of the prevalence of water in biological systems.

Photochemical transformations proceed either through resonant absorptionof one or more photons to a discrete molecular state, or throughnon-resonant absorption to a broad molecular band. Most relevantresonant transitions take place in the infrared (for vibrationaltransitions) to the visible (for electronic transitions). Most relevantresonant transitions depend so strongly on the frequency of light,however, that they are highly unlikely to be driven by the monochromaticlight from any particular infrared or visible laser. Transitions tobroad bands take place mostly in the ultraviolet end of the opticalspectrum and so should not be driven either by infrared light or byvisible light.

“Opticution” (described hereinbefore) is believed to be drivenprincipally by photochemistry, rather than by heating or mechanicaldisruption, and thus it is important to understand why visible (orultraviolet in some cases) optical tweezers can result in deleteriouseffects on biological and other materials having similar photochemistryresponses (or other optically driven events which lead to deleteriouseffects in any type of material, such as light sensitive chemicalstates, light sensitive electronic states or even sensitive mechanicalstructures at the microscopic level). Such materials can include, forexample, small molecule drugs, doped semiconductors, high temperaturesuperconductors, catalysts and low melting point metals.

For example, living organisms' resistance to photochemical degradationat visible wavelengths would appear to be a natural byproduct of theirevolution in sunlight. However, the flux of visible light from the sunis smaller than that in a typical 1 mW optical tweezer by some sixorders of magnitude. The intense illumination at the focal spot of anoptical tweezer greatly increases the rate of multiple-photon absorptionin which two or more photons cooperate to drive a single opticaltransition. Multiple photon events require photons to arrivesimultaneously, and so the level of occurrences of such events dependstrongly on the light intensity. The strong focus of an optical trapdoes indeed provide the high-intensity light environment needed to drivesuch multiple photon processes.

Multiphoton absorption can be more damaging in visible tweezers than ininfrared due to the simultaneous absorption of two visible photons whichdelivers the equivalent energy of a single ultraviolet photon. Likewisethis can be extended to ultraviolet photons having wavelengths in whichtwo or more such photons are required to deliver light energy whichwould alter the chemical, biological, electronic or mechanical state ofa material. Two-photon absorption of infrared light, on the other hand,delivers the equivalent energy of a visible photon and therefore doesnot usually suffice to drive photochemical or like opticaltransformations. Achieving relevant photochemistry events with infraredlight would therefore require three—or even four-photon absorption.Because higher-order absorption processes are less likely than lowerorder processes, visible optical tweezers appear to be more likely thaninfrared tweezers to induce deleterious photochemistry, such aschromosome recombinations in biological materials. This is believed tobe the mechanism by which tightly focused pulses of light at λ=532 nmform an optical scalpel capable of precisely cutting chromosomes.

The approximate rate W_(n) of an n-photon absorption in the focal volumeof an optical tweezer should scale as follows:${W_{n} \propto \left( {\frac{P}{\hslash \quad c}\quad \frac{\sigma}{\lambda}} \right)^{n}},$

where P is the power in the beam and σ is the photon capturecross-section for the absorber. As is shown by the above equation,lower-order processes at shorter wavelengths occur much more frequentlythan higher-order processes at longer wavelengths, at least for beams ofequal power.

Optical tweezers, however, are far more efficient when created withlight of shorter wavelengths than infrared light (e.g., visible and insome cases ultraviolet). As a result, such optical tweezers require muchless power to match the trapping force of infrared tweezers. Thiscomparative advantage of such tweezers opens the door to their use formicromanipulation of biological materials (and for any other highlylight sensitive materials as described hereinbefore). The magnitude ofthe optical gradient force drawing dielectric material to the focus ofan optical trap scales roughly with the inverse fourth power of λ in theRayleigh approximation: ${F \propto \frac{P}{\lambda^{4}}},$

Therefore a visible trap operating at, for example, λ=532 nm requiresonly {fraction (1/16)} the power to achieve the same trapping force asan infrared trap operating at λ=1064 nm. The relative reduction in powerimmediately translates into a reduction in the rate W₂ of two-photonabsorption events for the visible trap. Therefore, the likelihood ofsubstantial damage is drastically reduced and can even enable inflictionof virtually no damage to the subject material. Furthermore, by carefulselection of the wavelength of light used (visible or even ultravioletin some cases), absorption windows of a material being inspected can beused to select the wavelength of light to reduce the absorption of lightand thereby reduce the damage or alteration of the material.

The trapping efficiency of an optical tweezer can be increased stillfurther, and the power requirements correspondingly reduced, byappropriately shaping the wavefront of the trapping beam. For example,it has previously been demonstrated that an optical trap constructedfrom a donut-mode laser beam whose intensity vanishes at the opticalaxis requires far less power to achieve the axial trapping force of aconventional optical tweezer formed with a Gaussian TEM_(oo) mode. Whileit is clear that shaping the wavefront can improve trapping efficiency,thereby reducing two-photon absorption, no studies have reported anoptimal wavefront profile. Therefore, still further improvements areavailable with engineering of the trapping wavefront's characteristics.

While the time-averaged power establishes the trapping force of anoptical tweezer, its peak power sets the rate of occurrence oftwo-photon processes. Consequently, continuous-wave visible opticaltweezers should also be less damaging than traps derived from pulsedlasers.

The local irradiation, and thus the rate of two-photon processes, can bereduced still further by applying multiple separate traps to a system,as described hereinbefore, rather than just one trap. Distributing thetrapping force on an extended sample among N optical tweezers thereforereduces W₂ by a factor of N.

A system (biological or otherwise) with discrete photosensitivecomponents can be further trapped with visible (or ultraviolet in somecases) light tweezers, provided that care is taken to position the lighttraps away from sensitive areas of the subject material.

Further, some samples, such as many biological materials, absorb lightstrongly in the visible range and therefore cannot be trapped withvisible optical tweezers. For the great number of systems largelytransparent to visible light, various steps can be used to minimize therate of deleterious nonlinear optical processes. Without limitation,these can include:

1. Given a choice of a range of visible light wavelengths, one can avoidthe use of light wavelengths associated with strong absorptionscharacteristic of the type of material.

2. One can create traps with a continuous-wave (CW) laser, rather thanwith a pulsed laser.

3. One can trap an extended sample at multiple points, rather than atjust one.

4. One can make each trap as efficient as possible. For example, one cantake advantage of wavefront shaping to minimize the power required toachieve a desired trapping force.

The concepts of creating traps with a continuous-wave (CW) laser andavoiding those associated with strong absorptions can be appliedgenerically to all optical tweezer systems. Trapping extended samples atmultiple points and maximizing the efficiency of each trap, however,could be more difficult to implement in conventional optical tweezersystems, but are well within the domain of intended uses of holographicoptical tweezers. In particular, holographic optical tweezers (“HOTs”)can create an arbitrary number N of optical traps in arbitrary positionsso as to trap an extended biological sample (or other highly lightsensitive material) at multiple points. The simplest of these multipletrapping patterns also could be created by rapidly scanning a singletweezer among the desired array of traps. This can be particularlyuseful in biological samples, or other highly light sensitive materials,since not all of the power needs to be imparted on a single point of thesample. By instead distributing the power over a number of points, theoverall damage to the sample will be significantly reduced in a mannersimilar to a “bed of nails.” Achieving a desired time-averaged trappingforce in each of the N scanned traps, however, would require N times thepeak power in each trap. Consequently, HOTs have an inherent advantageover scanned tweezers when it comes to trapping materialsnondestructively.

HOTs also can produce more complex continuously evolving patterns oftraps than can scanned-tweezer systems. This would be an advantage ifoptical tweezer manipulation is intended to move or sort biological orhighly light sensitive materials.

Holographic optical tweezers also can tailor the wavefronts of theindividual beams making up the array of traps and can direct each beamaccurately into the trapping system's focusing optics. Consequently,optical tweezers formed with a HOT system can dynamically minimize theamount of power needed to achieve a desired trapping force.

The above qualitative example guidelines can be used for minimizing therate of radiation or other light initiated damage inflicted onbiological systems (or other highly light sensitive materials) byoptical trapping with visible light. By following these guidelines, itis possible to obtain acceptable small rate of damage for a particularapplication. The use of visible light in an HOT system would thereforehave at least several advantages for applications to biological systems:

Optical efficiency. Microscope objective lenses suitable for formingoptical tweezers typically are optimized for use at visible andultraviolet wavelengths and suffer from a variety of defects when usedto transmit infrared light. Trapping with visible light thus takesoptimal advantage of the designed properties of conventional optics.Infrared systems, by contrast either must use more costlyspecial-purpose optics, or else will suffer from optical aberrationswhich will somewhat diminish their potential benefits relative tovisible trapping systems.

Safety. The human visual system includes a protective blink reflex whichreduces the chance that a stray beam of light from a visible trappingsystem could damage a user's eyesight. No such reflex protects a user'svision in the infrared.

Ease of alignment. Visible optical trains are much easier to align thaninfrared.

Availability of two-photon processes. Two-photon processes can be usefulin some biological applications, for instance in creating opticalscissors and scalpels. The same optical train which createsdamage-minimizing configurations of visible optical traps can bereconfigured in real time to produce individual beams optimized fortwo-photon absorption, but with maximum power usage efficiency. Thus thesame system could trap, cut, and generally induce photochemicaltransformations in its samples using a single laser for excitation tofor one or a matrix of optical traps.

Reduced heating. Infrared systems probably heat their samples throughdirect absorption by water to a greater extent than do visible systems.This excess heating could explain some of the damage reported ininfrared trapping experiments on living systems.

Improved trapping accuracy. The trapping volume of an optical tweezerscales with the wavelength of light. Visible light therefore can trapmore accurately localized regions than infrared.

Improved tracking accuracy. Optical traps sometimes are used to trackthe motions of trapped objects, for instance through the time evolutionof the light scattered by trapped particles into the far field. Theresolution of such tracking techniques scales with the wavelength oflight and thus would be improved with traps formed in visible lightrelative to infrared.

It is also possible that a variety of biological and also nonbiologicalmaterials can be manipulated in a manner described above with varyingwavelengths, laser types, and experimental conditions. The particularparameters that are used will be dependent upon the material to bemanipulated and the optical, chemical, mechanical and electrical stateswhich are sensitive to light. More particularly for example, theabsorption characteristics of the material at certain wavelengths in thevisible range may have a significant bearing on the wavelength of thelaser beam used for the manipulation. For example, the use of certaintypes of green laser light can be successfully used with certainmaterials but not with others (such as chloroplasts in certain types ofplants). For nonbiological materials, such as electronic devices, onecan select visible light wavelengths which do not exhibit strongabsorption by the components of the device.

It is also noted that the visible portion of the spectrum has often beenconsidered to be in the range of about 400 nm to about 700 nm. It ispossible, however, that a broader wavelength range could be used inaccordance with the broader aspects of the invention. For example, thewindow of transparency for water is between about 200 nm and about 800nm, and an even larger range could be used in certain situations.

The following non-limiting examples demonstrate the efficacy of visibleoptical tweezers for manipulating living biological samples. Inparticular, we have demonstrated long-term trapping using light at λ=532nm using a frequency-doubled Nd:YV O₄ laser.

EXAMPLE I

Large numbers of yeast cells (generic varieties from a package ofFleischman's Yeast) have been trapped in culture medium and haveobserved several generations of budding during continuous illumination.During these tests, light having a wavelength of 532 nm from afrequency-doubled Nd:YV O₄ laser was used to trap the yeast cells. Theyeast included various strains of S. cerevisiae in aqueous solution atroom temperature. Individual cells were trapped with about 1 mW ofcontinuous-wave laser light. In one demonstration, sixteen cells wereconfined to a four by four array. Of these sixteen cells, about halfappeared to be budding daughter cells and forming colonies after sixhours.

EXAMPLE II

Light having a wavelength of 532 nm from a frequency-doubled Nd:YV O₄laser was used to trap a plurality of cheek epithelial cells. Swabbedcells were suspended in aqueous solution at room temperature anddeposited onto glass cover slips. Optical tweezers were trained on thenuclei and vacuoles of various cells for up to ten minutes. When regionsof the cell membrane were trapped strongly enough to displace asuspended cell through its culture medium, its internal processes didnot appear to be significantly, as determined by visual inspection.Normal cell function appeared to resume after the tweezers wereextinguished.

EXAMPLE III

Light having a wavelength of 532 nm from a frequency-doubled Nd:YV O₄laser was used to trap wheat chancre cells. The cells were obtained insolid medium and deposited onto glass cover slips before opticaltrapping at room temperature. Continuous optical trapping was notsufficient to disrupt the cell wall. Visual inspection of illuminatedcells provides qualitative results similar to those obtained with cheekepithelial cells.

While preferred embodiments of the invention have been shown anddescribed, it will be clear to those skilled in the art that variouschanges and modifications can be made without departing from theinvention in its broader aspects as set forth in the claims providedhereinafter.

What is claimed is:
 1. A method of manipulating a biological materialusing a focused beam of laser light, comprising the steps of: providinga focused beam of laser light with a wavelength selected from thevisible and ultraviolet wavelength range to form conditions for anoptical trap, the laser light having the wavelength selected from thevisible and ultraviolet wavelength range such that the selected materialexhibits a selected absorption coefficient in the wavelength selectedfrom the visible and ultraviolet wavelength range of the laser lightwhich permits manipulation without substantial damage to the biologicalmaterial; providing a plurality of the optical traps with the focusedbeam of laser light in the visible and ultraviolet wavelength range, theplurality of optical traps manipulating the biological material; andcontrolling the power level of each of the optical traps, the laserlight in the visible and ultraviolet wavelength range and the selectedresulting absorption coefficient to avoid substantial damage to thebiological material while manipulating the biological material.
 2. Themethod of claim 1, wherein the focused beam of laser light includeslight in the wavelength of about 400 nm to about 700 nm.
 3. The methodof claim 1, wherein the beam of light comprises a continuous-wave laserbeam.
 4. The method of claim 1, further comprising the step of shapingthe wavefront of the focused beam of light such that the power requiredto achieve a predetermined trapping force is reduced relative to afocused beam of light that has not been shaped.
 5. The method of claim4, wherein the beam of light comprises a donut mode.
 6. The method ofclaim 1, wherein the plurality of optical traps arise from a diffractiveoptical element.
 7. The method of claim 1, wherein the power level ofeach of the plurality of optical traps is controlled to avoid alteringthe genetic code by a biologically significant amount of the biologicalmaterial.
 8. A method of manipulating a biological material using afocused beam of laser light, comprising the steps of: providing acontinuous-wave, focused beam of laser light having a wavelengthselected from the visible and ultraviolet wavelength range such that thematerial possesses a weak absorption coefficient in the visible andultraviolet range wavelength of the laser light; providing at least oneoptical trap for manipulating a discrete portion of the biologicalmaterial; and controlling the power level of the at least one opticaltraps in conjunction with the weak absorption coefficient in the visibleand ultraviolet wavelength range to avoid alteration of a substantialportion of genetic code of the biological material during manipulatingthe biological material.
 9. The method of claim 8, wherein a pluralityof the optical traps are formed with the focused beam of laser light,wherein each of the traps manipulates a discrete portion of thematerial.
 10. The method of claim 9, further comprising the step ofshaping the wavefront of the focused beam of light such that thetrapping efficiency of the at least one optical trap is increasedrelative to not shaping the focused beam of light.
 11. The method ofclaim 10, wherein the focused beam of light comprises a shape calculatedto minimize absorption by the material.
 12. The method of claim 9,wherein each trapping location is not at a biologically sensitivelocation in the biological material.
 13. A system for manipulating abiological material using a focused beam of laser light, comprising: afocused beam of laser light having a wavelength selected from thevisible and ultraviolet range wherein the maximum wavelength is lessthan the minimum wavelength of infrared light, the focused laser lightforming an optical trap and having a wavelength such that the biologicalmaterial possesses a weak absorption coefficient in the visible andultraviolet wavelength range of the laser light used to manipulate thebiological material; and a plurality of the optical traps formanipulating discrete portions of the biological material, wherein thepower level of each of the optical traps is controlled and along withthe weak absorption coefficient of the biological material for the laserlight in the range selected from the visible and ultraviolet range whichenables avoiding at least the alteration of the genetic code of abiologically significant mount of the biological material.
 14. Thesystem of claim 13, wherein the focused beam of laser light is selectedfrom the visible and ultraviolet wavelength range.
 15. The system ofclaim 13, wherein the focused beam of laser light comprises acontinuous-wave laser beam.
 16. The system of claim 15, wherein thewavefront of the focused beam of laser light shaped to achieve apredetermined trapping force is reduced relative to a focused beam oflight that has not been shaped.
 17. The system of claim 13, wherein theplurality of optical traps comprise holographic optical traps.
 18. Amethod of manipulating a biological material using a focused beam oflaser light, comprising the steps of: providing a focused beam of laserlight to form conditions for an optical trap, the laser light having awavelength selected from the visible and ultraviolet range such that theselected material exhibits a selected absorption coefficient for thewavelength selected from the visible and ultraviolet range of the laserlight which permits manipulation without substantial damage to thebiological material; providing a plurality of the optical traps with thefocused beam of laser light selected from the visible and ultravioletrange, the plurality of optical traps manipulating the biologicalmaterial; and controlling the power level of each of the optical traps,the laser light wavelength selected from the visible and ultravioletrange and the selected resulting absorption coefficient to avoidsubstantial damage to the biological material while manipulating thebiological material.